Microtubules spatial alterations in root cells of Brassica rapa under clinorotation

Organization of tubulin cytoskeleton in epidermis and cortex cells in different root growth zones in Brassica rapa L. 6-day-old seedlings under clinorotation has been investigated. It was shown that changes in cortical microtubules orientation occur only in the distal elongation zone. In control, cortical microtubule arrays oriented transversely to the root long axis. Whereas under clinorotation an appearance of shorter randomly organized cortical microtubules was observed. Simultaneously, a significant decrease in a cell length in the central elongation zone under clinorotation was revealed. It is suggested that the decline of anisotropic growth, typical for central elongation zone cells, is connected with cortical microtubules disorientation under clinorotation.     

Interaction of microtubules and microfilaments in the zone of distal elongation of Arabidopsis thaliana roots

Investigations on GFP-MAP4 and GFP-ABD2 plants of Arabidopsis thaliana with the usage of inhibitors of actin and tubulin polymerization revealed that stability of cell growth in the zone of distal elongation of a root is provided by structural interactions between microtubules and actin filaments. The experiments with clinorotation and inhibition analysis enabled us to demonstrate that gravitation is a determining factor for the interaction between cytoskeleton elements in the zone of distal elongation in a root.     

Microtubules in epidermal and cortical root cells of Brassica rapa during clinorotation

Using confocal microscopy the organization of tubulin cytoskeleton including endoplasmic and cortical microtubules (CMTs) has been studied in epidermal and cortical cells of the different growth zones of main root of Brassica rapa L. 6-days-old seedlings in control conditions and under clinorotation. It was shown that changes in CMTs orientation occured only in the distal elongation zone (DEZ). In the control, CMT arrays oriented transversely to the root long axis. Under clinorotation appearance of the shorter randomly organized CMTs was observed. Simultaneously, a significant decrease in the cell length in the central elongation zone (CEZ) under clinorotation was detected. It is suggested that the decline of anisotropic growth typical for CEZ cells is connected with CMTs disorientation under clinorotation.     

Hypersensitivity to cytoskeletal antagonists demonstrates microtubule-microfilament cross-talk in the control of root elongation in Arabidopsis thaliana

Elongation of diffusely expanding plant cells is thought to be mainly under the control of cortical microtubules. Drug treatments that disrupt actin microfilaments, however, can reduce elongation and induce radial swelling. To understand how microfilaments assist growth anisotropy, we explored their functional interactions with microtubules by measuring how microtubule disruption affects the sensitivity of cells to microfilament-targeted drugs. We assessed the sensitivity to actin-targeted drugs by measuring the lengths and diameters of expanding roots and by analysing microtubule and microfilament patterns in the temperature-sensitive Arabidopsis thaliana mutant microtubule organization 1 (mor1-1), along with other mutants that constitutively alter microtubule arrays. At the restrictive temperature of mor1-1, root expansion was hypersensitive to the microfilament-disrupting drugs latrunculin B and cytochalasin D, while immunofluorescence microscopy showed that low doses of latrunculin B exacerbated microtubule disruption. Root expansion studies also showed that the botero and spiral1 mutants were hypersensitive to latrunculin B. Hypersensitivity to actin-targeted drugs is a direct consequence of altered microtubule polymer status, demonstrating that cross-talk between microfilaments and microtubules is critical for regulating anisotropic cell expansion.     

Ultrastructure of statocytes and cells of distal elongation zone of Arabidopsis thaliana under clinorotation

Results of the electron-microscopic investigation of root apices of Arabidopsis thaliana 3-, 5- and 7-days-old seedlings grown in the stationary conditions and under clinorotation are presented. It was shown the similarity in the root apex cell ultrastructure in control and under clinorotation. At the same time there were some differences in the ultrastructure of statocytes and the distal elongation zone under clinorotation. For the first time the sensitivity of ER-bodies, which are derivatives of GER and contain beta-glucosidase, to the influence of simulated microgravity was demonstrated by increased quantity and area of ER-bodies at the cell section as well as by higher variability of their form under clinorotation. A degree of these changes correlated with the duration of clinorotation. On the basis of experimental data a protective role of ER-bodies in adaptation of plants to microgravity is supposed.     

Inhibition of phospholipase C disrupts cytoskeletal organization and gravitropic growth in Arabidopsis roots

The phospholipase protein superfamily plays an important role in hormonal signalling and cellular responses to environmental stimuli. There is also growing evidence for interactions between phospholipases and the cytoskeleton. In this report we used a pharmacological approach to investigate whether inhibiting a member of the phospholipase superfamily, phospholipase C (PLC), affects microtubules and actin microfilaments as well as root growth and morphology of Arabidopsis thaliana seedlings. Inhibiting PLC activity using the aminosteroid U73122 significantly inhibited root elongation and disrupted root morphology in a concentration-dependent manner, with the response being saturated at 5 μM, whereas the inactive analogue U73343 was ineffective. The primary root appeared to lose growth directionality accompanied by root waving and formation of curls. Immunolabelling of roots exposed to increasingly higher U73122 concentrations revealed that the normal transverse arrays of cortical microtubules in the elongation zone became progressively more disorganized or depolymerized, with the disorganization appearing within 1 h of incubation. Likewise, actin microfilament arrays also were disrupted. Inhibiting PLC using an alternative inhibitor, neomycin, caused similar disruptions to both cytoskeletal organization and root morphology. In seedlings gravistimulated by rotating the culture plates by 90°, both U73122 and neomycin disrupted the normal gravitropic growth of roots and etiolated hypocotyls. The effects of PLC inhibitors are therefore consistent with the notion that, as with phospholipases A and D, PLC likewise interacts with the cytoskeleton, alters growth morphology, and is involved in gravitropism.     

Cytoskeleton orientation in epidermis cells of roots generated de novo in leaf explants under clinorotation

The anatomy, cytoskeleton orientation, and thickness of the cell wall of the root growth zones generated de novo in vitro under clinorotation (simulated microgravity) were studied. The anatomical structure of the roots generated de novo from the cambium cells of the leaf explant petiole is similar to the structure of embryonic roots. The root cell differentiation in vitro during the clinorotation does not differ from the control in main features. Changes in the tubulin cytoskeleton orientation under clinorotation were detected in the epidermis of distal elongation zone (that is apparently associated with specific physiological properties of the cells in this zone). A tendency towards the thinning of the root cell walls in vitro under conditions of simulated microgravity was established.     

Alterations in the Cytoskeleton Accompany Aluminum-Induced Growth Inhibition and Morphological Changes in Primary Roots of Maize

Although Al is one of the major factors limiting crop production, the mechanisms of toxicity remain unknown. The growth inhibition and swelling of roots associated with Al exposure suggest that the cytoskeleton may be a target of Al toxicity. Using indirect immunofluorescence microscopy, microtubules and microfilaments in maize (Zea mays L.) roots were visualized and changes in their organization and stability correlated with the symptoms of Al toxicity. Growth studies showed that the site of Al toxicity was associated with the elongation zone. Within this region, Al resulted in a reorganization of microtubules in the inner cortex. However, the orientation of microtubules in the outer cortex and epidermis remained unchanged even after chronic symptoms of toxicity were manifest. Auxin-induced reorientation and cold-induced depolymerization of microtubules in the outer cortex were blocked by Al pretreatment. These results suggest that Al increased the stability of microtubules in these cells. The stabilizing effect of Al in the outer cortex coincided with growth inhibition. Reoriented microfilaments were also observed in Al-treated roots, and Al pretreatment minimized cytochalasin B-induced microfilament fragmentation. These data show that reorganization and stabilization of the cytoskeleton are closely associated with Al toxicity in maize roots.     

Microtubules and microfilaments are both responsible for pollen tube elongation in the coniferPicea abies (Norway spruce)

Summary InPicea abies (Norway spruce), microtubules and actin microfllaments both form a dense matrix throughout the tube mainly parallel to the direction of elongation. In these conifer pollen tubes the organization of this matrix is different from that in angiosperms. This study tests our hypothesis that differences in cytoskeletal organization are responsible for differences in tube growth and physiology. Pollen grains were germinated in media containing cytoskeletal disrupters and analyzed for germination, tube length, tube branching, and tip swelling. Disruption of microtubules significantly inhibits tube elongation and induces tube branching and tip swelling. Tip swelling is probably caused by disruption of the microtubules in the tip that are perpendicular to the direction of elongation. Confocal microscopy indicates that colchicine and propyzamide cause fragmentation of microtubules throughout the tube. Oryzalin and amiprophosmethyl cause a complete loss of microtubules from the tip back toward the tube midpoint but leave microtubules intact from the midpoint back to the grain. Disruption of microfilaments by cytochalasins B and D and inhibition of myosin by N-ethylmaleimide or 2,3-butanedione monoxime stops tube growth and inhibits germination. Microfilament disruption induces short branches in tubes, probably originating from defective microfilament organization behind the tip. In addition, confocal microscopy coupled with microinjection of fluorescein-labeled phalloidin into actively growing pollen tubes indicates that microfllament bundles extend into the plastid-free zone at the tip but are specifically excluded from the growing tip. We conclude that microtubules and microfilaments coordinate to drive tip extension in conifer pollen tubes in a model that differs from angiosperms.     

Ultrastructure of the statocytes and cells of the distal elongation zone of <Emphasis Type="Italic">Arabidopsis Thaliana</Emphasis> under the conditions of clinorotation

This paper presents the results of an electron microscope study of the root apices of 3-, 5- and 7- day-old seedlings of Arabidopsis thaliana grown under the conditions of stationary control and clinorotation. We demonstrated both the similarities with the control group and the differences in the ultrastructure of statocytes and cells of the distal zone of elongation under clinorotation. For the first time it was possible to establish the sensitivity of ER bodies, which are granular endoplasmic reticulum derived organelles containing β-glucosidase, to the influence of simulated microgravity. As a result, an increase in the number and area of ER bodies per section of a cell and in the variability of their forms was detected. The degree of these changes correlated with the duration of clinorotation. The supposition concerning the protective role of ER bodies in plant adaptation to microgravity is based on the data obtained.     

Microtubule reorganization as a response to implementation of NO signals in plant cells

The effects of an exogeneous NO donor, sodium nitroprusside, on the orientation and organization of cortical microtubules in Arabidopsis thaliana root cells expressing GFP-MAP4 were studied in vivo. It was found that sodium nitroprusside treatment (10–500 μM, 24 h) caused the acceleration of primary root growth and enhanced initiation of root hairs in the differentiation zone. The influence of sodium nitroprusside revealed in changes in the orientation and organization of cortical microtubules in different types of cells of A. thaliana root. The most sensitive to sodium nitroprusside exposure were microtubules in epidermal cells of the elongation zone, where native transverse orientation of cortical microtubules turned into random, oblique, or longitudinal relative to the primary root axis. We suggest that NO, as one of the intracellular secondary messengers, triggers cell differentiation by reorientation of cortical microtubules, possibly via tubulin nitrotyrosination.     

Influence of cold on organization of actin filaments of different types of root cells in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The effect of the low temperature (+4°C) on the organization of actin filaments (microfilaments) of cells from different growth zones has been studied in the roots of Arabidopsis thaliana (L.). It was found that cold treatment inhibited the growth of the primary root and changed its morphology, causing a formation of large number of deformed (ectopic) root hairs in differentiation zone. The temporal relationship between the disorientation and the organization of actin filaments and the detected changes of growth and morphology of roots after cold treatment was shown. It has been found that actin filaments of root hairs, meristematic cells, cells of elongation zone, and epidermal cells of all root zones of A. thaliana are the most sensitive to the cold.     

Growth and differentiation of the root cap columella and root proper under stationary conditions and in clinorotation

Results are presented from light-optical and electron-microscopy studies of the root apices of three-day-old seedlings of table beet (Beta vulgaris) grown under conditions of stationary control and in clinorotation. It is shown that the ultrastructure and topography of the organelles of the root cap statocytes (gravity-perceptive cells) and cells of the distal elongation zone (gravity-receptor cells) of the root proper clearly reflect different directions for their growth and differentiation in space and in time as a function of specialization and function. Growth and genetically determined differentiation of the cells under clinorotation conditions occur as in the control, though certain differences in their ultrastructure attest to changes in metabolism.     

Tissue slices from living root caps as a model system in which to study cytodifferentiation of polar cells

Tissue slices of living root caps of cress (Lepidium sativum L.), two to three cell layers in thickness, were prepared by a microsurgical procedure. The viability, cellular structures and cytoplasmic movement of the cells were examined in the light microscope. Nuclei, amyloplasts, vacuoles and endoplasmic reticulum were identified and their positions confirmed after fixation and observation of the same cells in the electron microscope. The distribution of microtubules was shown by immunocytochemistry. During germination, microtubules appear first at the distal edges of the statocytes, while in mature statocytes a distal domain of criss-crossed microtubules could be distinguished from a proximal domain with transversally oriented microtubules. Microfilaments in young statocytes form a nuclear enclosure; in mature statocytes bundles of microfilaments fan out into the cell cortex. The transition from statocytes to secretion cells is accompanied by a more pronounced cortical network of microfilaments, while the nucleus-associated microfilaments remain visible. It is suggested that these microfilaments play a role in the positioning of the nucleus and the translocation of endoplasmic reticulum.Abbreviations ER endoplasmic reticulum - MF microfilament - MT microtubule     

Microtubules regulate aortic endothelial cell actin microfilament reorganization in intact and repairing monolayers

To understand the role of microtubules and microfilaments in regulating endothelial monolayer integrity and repair, and since microtubules and microfilaments show some co-alignment in endothelial cells, we tested the hypothesis that microtubules organize microfilament distribution. Disruption of microtubules with colchicine in resting confluent aortic endothelial monolayers resulted in disruption of microfilament distribution with a loss of dense peripheral bands, an increase in actin microfilament bundles, and an associated increase of focal adhesion proteins at the periphery of the cells. However, when microfilaments were disrupted with cytochalasin B, microtubule distribution did not change. During the early stages of wound repair of aortic endothelial monolayers, microtubules and microfilaments undergo a sequential series of changes in distribution prior to cell migration. They are initially distributed randomly relative to the wound edge, then align parallel to the wound edge and then elongate perpendicular to the wound edge. When microtubules in wounded cultures were disrupted, dense peripheral bands and lamellipodia formation were lost with increases in central stress fibers. However, following microfilament disruption, microtubule redistribution was not disrupted and the microtubules elongated perpendicular to the wound edge similar to non-treated cultures. Microtubules may organize independently of microfilaments while microfilaments require microtubules to maintain normal organization in confluent and repairing aortic endothelial monolayers.     

The effect of okadaic acid on Arabidopsis thaliana root morphology and microtubule organization in its cells

To investigate the role of protein hyperphosphorylation in plant cells, the effect of okadaic acid, a specific inhibitor of protein phosphatases PPI and PP2A, on the general morphology of Arabidopsis thaliana primary roots and the structural-functional characteristics of cortical microtubules in different cell types in all primary root growth zones was studied. It was found that okadaic acid affects microtubule organization in a different manner depending on the type of cells and functional zones of the primary root. Cortical microtubules in the epidermis and cortex cells of the elongation zone proved to be most sensitive to 0.1, 1, and 10 nM okadaic acid which completely depolymerized after inhibitor treatment. In trichoblasts, atrichoblasts of differentiation zone treatment with okadaic acid caused the microtubules stabilization. The treatment with okadaic acid significantly affected the morphology of root hairs, causing their swelling and branching as a result of abnormal microtubule orientation. The results of this study suggest that induction of protein hyperphosphorylation as a result of protein phosphatase inhibition plays a crucial key in microtubule organization in plant cells.     

Polarized cell growth, organelle motility, and cytoskeletal organization in conifer pollen tube tips are regulated by KCBP, the calmodulin-binding kinesin

Kinesin-like calmodulin-binding protein (KCBP), a member of the Kinesin 14 family, is a minus end directed C-terminal motor unique to plants and green algae. Its motor activity is negatively regulated by calcium/calmodulin binding, and its tail region contains a secondary microtubule-binding site. It has been identified but not functionally characterized in the conifer Picea abies. Conifer pollen tubes exhibit polarized growth as organelles move into the tip in an unusual fountain pattern directed by microfilaments but uniquely organized by microtubules. We demonstrate here that PaKCBP and calmodulin regulate elongation and motility. PaKCBP is a 140 kDa protein immunolocalized to the elongating tip, coincident with microtubules. This localization is lost when microtubules are disrupted with oryzalin, which also reorganizes microfilaments into bundles. Colocalization of PaKCBP along microtubules is enhanced when microfilaments are disrupted with latrunculin B, which also disrupts the fine network of microtubules throughout the tip while preserving thicker microtubule bundles. Calmodulin inhibition by W-12 perfusion reversibly slows pollen tube elongation, alters organelle motility, promotes microfilament bundling, and microtubule bundling coincident with increased PaKCBP localization. The constitutive activation of PaKCBP by microinjection of an antibody that displaces calcium/calmodulin and activates microtubule bundling repositions vacuoles in the tip before rapidly stopping organelle streaming and pollen tube elongation. We propose that PaKCBP is one of the target proteins in conifer pollen modulated by calmodulin inhibition leading to microtubule bundling, which alters microtubule and microfilament organization, repositions vacuoles and slows organelle motility and pollen tube elongation.     

The growth and differentiation of root cap columella cells and the proper root grown in the stationary conditions and under clinorotation

The results of light- and electron-microscopic investigations of root apices of Beta vulgaris 3-day-old seedlings grown in the stationary conditions and under clinorotation are presented. It was shown that ultrastructure and topography of organelles in root cap statocytes (graviperceptive cells) and in the cells of distal elongation zone clearly reflected the different direction in their growth and differentiation in space and time in dependence on specialization and functions. Cell growth and genetically determined differentiation occur similarly to control, although certain differences in ultrastructure are evident on metabolism changes.     

Microtubules and microfilaments coordinate to direct a fountain streaming pattern in elongating conifer pollen tube tips

This study investigates how microtubules and microfilaments control organelle motility within the tips of conifer pollen tubes. Organelles in the 30-m-long clear zone at the tip of Picea abies (L.) Karst. (Pinaceae) pollen tubes move in a fountain pattern. Within the center of the tube, organelles move into the tip along clearly defined paths, move randomly at the apex, and then move away from the tip beneath the plasma membrane. This pattern coincides with microtubule and microfilament organization and is the opposite of the reverse fountain seen in angiosperm pollen tubes. Application of latrunculin B, which disrupts microfilaments, completely stops growth and reduces organelle motility to Brownian motion. The clear zone at the tip remains intact but fills with thin tubules of endoplasmic reticulum. Applications of amiprophosmethyl, propyzamide or oryzalin, which all disrupt microtubules, stop growth, alter organelle motility within the tip, and alter the organization of actin microfilaments. Amiprophosmethyl inhibits organelle streaming and collapses the clear zone of vesicles at the extreme tip together with the disruption of microfilaments leading into the tip, leaving the plasma membrane intact. Propyzamide and oryzalin cause the accumulation of membrane tubules or vacuoles in the tip that reverse direction and stream in a reverse fountain. The microtubule disruption caused by propyzamide and oryzalin also reorganizes microfilaments from a fibrillar network into pronounced bundles in the tip cytoplasm. We conclude that microtubules control the positioning of organelles into and within the tip and influence the direction of streaming by mediating microfilament organization.Electronic Supplementary Material Supplementary material is available in the online version of this article at Abbreviations APM Amiprophosmethyl - FITC Fluorescein isothiocyanate - LATB Latrunculin B     

The Type II Arabidopsis Formin14 Interacts with Microtubules and Microfilaments to Regulate Cell Division

Formins have long been known to regulate microfilaments but have also recently been shown to associate with microtubules. In this study, Arabidopsis thaliana FORMIN14 (AFH14), a type II formin, was found to regulate both microtubule and microfilament arrays. AFH14 expressed in BY-2 cells was shown to decorate preprophase bands, spindles, and phragmoplasts and to induce coalignment of microtubules with microfilaments. These effects perturbed the process of cell division. Localization of AFH14 to microtubule-based structures was confirmed in Arabidopsis suspension cells. Knockdown of AFH14 in mitotic cells altered interactions between microtubules and microfilaments, resulting in the formation of an abnormal mitotic apparatus. In Arabidopsis afh14 T-DNA insertion mutants, microtubule arrays displayed abnormalities during the meiosis-associated process of microspore formation, which corresponded to altered phenotypes during tetrad formation. In vitro biochemical experiments showed that AFH14 bound directly to either microtubules or microfilaments and that the FH2 domain was essential for cytoskeleton binding and bundling. However, in the presence of both microtubules and microfilaments, AFH14 promoted interactions between microtubules and microfilaments. These results demonstrate that AFH14 is a unique plant formin that functions as a linking protein between microtubules and microfilaments and thus plays important roles in the process of plant cell division.